Arachidonic acid is an unsaturated fatty acid that is a precursor in the synthesis of compounds, such as prostaglandins, hydroxy-acids and leukotrines, that are involved in inflammation reactions. It is released from membrane phospholipids by phospholipase A.sub.2 activity. In response to anti-inflammatory agents, such as glucocorticoids, certain cells release proteins that have been characterized in vitro by their ability to inhibit phospholipase A.sub.2. Accordingly, by inhibiting arachidonic acid production, lipocortins block the synthesis of prostaglandins and other inflammatory substances, thereby reducing inflammation [F. Hirata et al., "A Phospholipase A.sub.2 Inhibitory Protein In Rabbit Neutrophils Induced By Glucocorticoids", Proc. Natl. Acad. Sci. U.S.A., 77, No. 5, pp. 2533-36 (1980)].
To date, several phospholipase A.sub.2 inhibitory proteins have been studied. One of them--lipomodulin--has been characterized as an about 40,000 molecular weight protein that is probably degraded by proteases in the cell to two smaller active species of about 30,000 and 15,000 molecular weight [F. Hirata et al., "Identification Of Several Species Of Phospholipase Inhibitory Protein(s) By Radioimmunoassay For Lipomodulin", Biochem. Biophys. Res. Commun., 109, No. 1, pp. 223-30 (1982)]. Other experimental evidence suggests that two other phospholipase A.sub.2 inhibitors, macrocortin (about 15,000 molecular weight) and renocortin (two species with molecular weights of about 15,000 and 30,000 respectively) may also be cleavage products of larger inhibitory proteins such as lipomodulin [J. F. Cloix et al., "Characterization And Partial Purification Of Renocortins: Two Polypeptides Formed In Renal Cells Causing The Anti-Phospholipase-like Action Of Glucocorticoids", Br. J. Pharmac., 79. pp. 313-21 (1983); G. J. Blackwell et al., "Macrocortin: A Polypeptide Causing The Anti-Phospholipase Effect Of Glucocorticoids", Nature, 287, pp. 147-49 (1980)].
Although lipomodulin has been isolated from rabbit neutrophil cells, macrocortin from rat macrophages and renocortin from rat renomedullary interstitial cells, the three proteins exhibit similar biological activities, molecular weights and cross-reactivity with monoclonal antibodies against lipomodulin or macrocortin. Moreover, all are induced by glucocorticoids. Thus, it has been suggested that these phospholipase inhibitory proteins, or lipocortins, are closely related to each other and are produced by cells as a general physiological mechanism of steroid action [B. Rothhut et al., "Further Characterization Of The Glucocorticoid-Induced Antiphospholipase Protein `Renocortin`", Biochem. Biophys. Res. Commun., 117, No. 3, pp. 878-84 (1983)].
Recent data have also indicated that the 15,000 molecular weight species of lipomodulin is produced by lymphocytes in response to immunogens and acts as a glycosylation-inhibiting factor, inhibiting the glycosylation of IgE-binding factors and leading to the suppression of the IgE response [T. Uede et al., "Modulation Of The Biologic Activities Of IgE-Binding Factors: I. Identification Of Glycosylation-Inhibitory Factor As A Fragment Of Lipomodulin", J. Immunol., 130, No. 2, pp. 878-84 (1983)].
As a result of their anti-inflammatory activities, lipocortins are useful for the treatment of disorders involving inflammatory processes. Such disorders include arthritic, allergic, dermatologic, ophthalmic and collagen diseases. Furthermore, the use of these proteins to treat inflammation might avoid the disadvantages now associated with present anti-inflammatory compounds.
At present two classes of compounds are being used for anti-inflammatory therapy: corticosteroids and nonsteroidal anti-inflammatory drugs. Corticosteroids are generally disfavored due to the severe side effects that may be associated with their use. These effects include hypertension, gastrointestinal bleeding, muscle weakness, cataracts and convulsions. Thus, nonsteroidal anti-inflammatory compounds are preferred. However, these non-steroids may also produce side effects, such as adverse effects on gastric and platelet physiology and on the central nervous system and hematopoiesis. In addition, most non-steroidal anti-inflammatory agents inhibit the production of inflammatory substances via their effect on only one of the two pathways for production of those substances, i.e., either the cyclooxygenase pathway or the lipoxygenase pathway.
In contrast, lipocortins inhibit the production of inflammatory substances via both pathways. Furthermore, because lipocortins are only mediators of steroid action, it is unlikely that they will produce the side effects often associated with the use of corticosteroids. And because these inhibitor proteins are natural mediators produced by the cell, they are unlikely to have the side effects usually associated with many non-steroid anti-inflammatories.
In addition, lipocortin may affect inflammation through an alternate pathway by blocking the chemotactic response of leukocytes. It has been shown that certain peptides whose sequences code for the tyrosine phosphorylation sites found within pp60 src and MT antigen, i.e., the src peptide and the MT peptide, inhibit chemotaxis of rabbit neutrophils stimulated by a chemoattractant [F. Hirata et al., "Inhibition Of Leukocyte Chemotaxis By Glu-Glu-Glu-Glu-Tyr-Pro-Met-Glu And Leu-Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg-Gln-Gly", Biochem. Biophys. Res. Comm., 18, pp. 682-90 (1984)]. Lipocortin contains a similar sequence within its amino acid sequence (GluAsnGluGluGlnGluTyr, residue number 15-21). Therefore, lipocortin may exert its anti-inflammatory effect via this sequence by inhibiting or blocking the movement of neutrophils and macrophages into inflammed tissue.
To date, however, human lipocortins have not been purified from cells. Furthermore, even if a procedure could be developed for the purification of lipocortins, it is doubtful that sufficient quantities of them could be produced for their many clinical and commercial applications. Accordingly, processes enabling the production of human lipocortins in clinically useful amounts would be highly advantageous in anti-inflammatory therapy.